Project Summary/Abstract The NuRD Complex Regulates ZEB1 Dimerization and NSCLC Metastasis Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related death in the United States, due in part to the robust affinity of lung cancer cells to metastasize. Understanding the processes that contribute to metastasis provides promise for the discovery of novel therapeutic targets. Epithelial-to-mesenchymal transition (EMT) is a proposed model for the initiation of metastasis. During EMT cell adhesion and polarity is reduced, allowing epithelial cancer cells to dissociate from the primary tumor and invade distant organs. The transcription factor zinc finger E-box-binding homeobox 1 (ZEB1) has been reported to uniquely correlate with NSCLC disease progression and to confer therapy resistance in multiple tumor types. Additionally, depletion of ZEB1 has been found to reverse therapy resistance, hence uncovering regulators of ZEB1 provides promise for innovative therapeutic strategies that may improve lung cancer patient outcome. Recent publications demonstrate that ZEB1 undergoes post-translational modification, suggesting a method for regulating ZEB1 function; however, the extent to which ZEB1 is modified as well as the purpose of ZEB1 modification has not been fully elucidated. We have applied the BioID screening method, which exploits a promiscuous biotin ligase, BirA, for proximity-dependent biotinylation of proteins vicinal to ZEB1. BioID methodology allowed us to uncover stable, weak, and transient ZEB1 interactors, which may regulate ZEB1 function and post-translational modifications. This screen revealed an interaction amongst ZEB1 and the histone deacetylase (HDAC)- containing nucleosome and deacetylase remodeling complex (NuRD). Through treatment with class I HDAC inhibitors Trichostatin A or Mocetinostat, we were able to induce a >100 kDa molecular weight shift. We have subsequently identified this as an SDS-PAGE resistant homodimer, suggesting that class I HDACs regulate ZEB1 dimerization by post-translational modification. Furthermore, we confirmed that the NuRD complex is a ZEB1 co-repressor, contributing to ZEB1 induced EMT. Consequently, we sought to define potential ZEB1- NuRD target genes that contribute to NSCLC disease progression. Through analysis of chromatin immunoprecipiation-sequencing (chIP-seq) data provided by the ENCODE project (ENCyclopedia Of DNA Elements), we identified EPS8L2, TBC1D2a and TBC1D2b as ZEB1-NuRD target genes. Intriguingly, these genes have been described in autophagy suppression; however, the significance of these targets in metastatic NSCLC has not been identified. The goal of proposal will be to provide insight to the role of ZEB1 post- translational modification on dimerization and autophagy in metastatic NSCLC.